Endothelial cell tropic compositions and methods of making and using the same

ABSTRACT

An isolated nucleic acid molecule comprising a modified Murine Leukemia Virus Long Terminal Repeat sequence that is capable of tropically regulating expression of operably linked coding sequences in a vessel endothelial cell is disclosed. A method of producing a non-Murine Leukemia Virus protein in a vessel endothelial cell is disclosed. The method comprises the step of introducing into the cell a nucleic acid molecule that comprises a modified Murine Leukemia Virus Long Terminal Repeat sequences operably linked to a nucleotide sequence which encodes a non-Murine Leukemia Virus protein, the modified Murine Leukemia Virus Long Terminal Repeat sequences being capable of tropically regulating expression in vessel endothelial cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 filing of PCT/US94/1088 filed Sep. 9, 1994which is a continuation-in-part of U.S. application Ser. No. 08/121,051filed Sep. 10, 1993, now abandoned, the disclosure of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to genetic regulatory sequences thatdirect vessel endothelial cell specific expression of genes, chimericgenes comprising the regulatory sequence linked to coding sequences andto methods of producing proteins in endothelial cells using the chimericgenes.

BACKGROUND OF THE INVENTION

Advances in molecular biology over the past few decades have created newtechnologies for combating human disease. In particular, thetransduction of foreign genes into somatic cells, or gene therapy, hasshown great promise as a powerful clinical tool. Gene therapy has beenused experimentally to correct disorders such as ADA deficiency and newtrials are underway for treating other genetic diseases such as cysticfibrosis and muscular dystrophy. Most gene therapy studies to date havefocused on correcting single gene defects by introducing the wild typegene into somatic cells. The product of the newly transduced gene thenrestores the normal phenotype. Other recent application includetargeting tumor cells with neo-antigens for enhanced recognition by theimmune system. Additionally, biologically active proteins may bedelivered to an individual by the introduction and expression of foreigngenes encoding such proteins.

Many studies have used bone marrow or bone marrow derived cells astargets for gene therapy. However, there has been a recent surge ofinterest in using other cell types, particularly endothelial cells.Endothelial cells represent an ideal target for gene therapy in manydiseases. Endothelial cells are particularly useful in the treatment ofhematological and vascular disorders as well as any disease thatrequires the systemic deliver of therapeutic factors.

Broader applicability of gene therapy has been hindered due to technicallimitations. Most studies thus performed have been so using infectedtissue ex vivo and then reconstituting the host with the geneticallyaltered cells. While this practice is acceptable for tissues that can beharvested and re-implanted with relative ease, many desired target cellsdo not fall into this category. For this reason, the search for and/orcreation of vectors that are expressed by specific tissues is an area ofintense research interest. There is a need for regulatory elements whichcan be used to direct expression of foreign genes and endothelial cells.There is a need to create tissue specific vectors for in vivo genetherapy. There is a need to provide gene therapy constructs withregulatory elements that will direct tissue specific expression of theforeign genes.

SUMMARY OF THE INVENTION

The present invention relates to isolated nucleic acid molecules thatcomprise a modified Murine Leukemia Virus Long Terminal Repeat whichcomprises SEQ ID NO:4. In some embodiments, such nucleic acid moleculesmay comprise SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.

The present invention relates to isolated nucleic acid molecules thatcomprise a modified Murine Leukemia Virus Long Terminal Repeat whichcomprises SEQ ID NO:4 and is operably linked to a nucleotide sequencethat encodes a non-Murine Leukemia Virus. In some embodiments, suchnucleic acid molecules may comprise SEQ ID NO:3, SEQ ID NO:2 or SEQ IDNO:1. In some embodiments, the non-Murine Leukemia Virus protein is ahuman protein, such as for example Factor VII protein, Factor IXprotein, von Willdebrand factor, complement proteins, insulin,cytokines, tissue plasminogen activator, alpha-L-iduronidase, iduronatesulfatase, heparin, N-sulfatase and alpha 1 antitrypsin.

The present invention relates to isolated nucleic acid molecules thatcomprise a modified Murine Leukemia Virus Long Terminal Repeat which: a)comprises SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1; b) isoperably linked to a nucleotide sequence that encodes a non-MurineLeukemia Virus and c) is encapsulated within a liposome or a viral coat.The viral coat may be that of an infectious, replicating viral particlesuch as a retrovirus or a viral coat of an infectious, non-replicatingviral package particle.

The present invention relates to methods of producing a non-MurineLeukemia Virus protein in an endothelial cell that comprises the step ofintroducing into said endothelial cell, nucleic acid molecule comprisinga modified Murine Leukemia Virus Long Terminal Repeat which comprisesSEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 operably linked toa nucleotide sequence that encodes a non-Murine Leukemia Virus. In someembodiments, the cell is a human vessel endothelial cell. In someembodiments, the protein is a human protein such as for example: FactorVII protein, Factor IX protein, von Willdebrand factor, complementproteins, insulin, cytokines, tissue plasminogen activator,alpha-L-iduronidase, iduronate sulfatase, heparin, N-sulfatase and alpha1 antitrypsin. In some embodiments, the nucleic acid molecule isencapsulated within a liposome or a viral coat. In some embodiments, thenucleic acid molecule is encapsulated within a viral coat of aninfectious, replicating viral particle such as retrovirus particle. Insome embodiments, the nucleic acid molecule is encapsulated within aviral coat of an infectious, non-replicating viral package particle. Insome embodiments, the human vessel endothelial cells are in anindividual. In some embodiments, the human vessel endothelial cells arein an individual and the nucleic acid molecule is deliveredintravenously to the individual. In some embodiments, the human vesselendothelial cells are in an individual, the nucleic acid molecule isdelivered intravenously to the individual and the nucleic acid moleculecomprises a modified Murine Leukemia Virus Long Terminal Repeat which:a) comprises SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1; b) isoperably linked to a nucleotide sequence that encodes a non-MurineLeukemia Virus and c) is encapsulated within a liposome or a viral coat.The viral coat may be that of an infectious, replicating viral particlesuch as a retrovirus or a viral coat of an infectious, non-replicatingviral package particle.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "tropism" is meant to refer to gene expressionwhich takes place in a cell specific manner. In particular, the term"endothelial cell tropism" is meant to refer to expression of codingsequences of nucleic acid molecules which occurs in endothelial cellsonly. Thus, the term "endothelial cell tropic compositions" refers tonucleic acid molecules which are specifically expressed in endothelialcells to the exclusion of other cells.

It has been discovered that modified forms of the long terminal repeat(LTR) of murine leukemia virus (MuLV) provides vessel endothelial celltropic gene expression. According to the invention, MuLV LTR sequenceswhich contain specific nucleotide sequences confer vessel endothelialcell tropism for expression of non-MuLV coding sequences operably linkedto such modified LTR sequences. When operably linked to the modifiedLTRs of the invention, nucleotide sequences that encode non-MuLVproteins will be expressed at a high level in vessel endothelial cellsbut not in other cell types.

In previously studied systems, MuLV LTRs that have been observed toregulate expression in endothelial do not do so exclusively ofexpression in other cell types. That is, endothelial cell expression isnot exclusive but rather is just one of many cell types in which theregulatory elements are functional. The present invention allows forgene expression specifically and exclusively in vessel endothelialcells. According to the present invention, modified MuLV LTRs of theinvention, such as the MuLV LTR molecular clone TR1.3, may be used toexpress non-MuLV proteins in vessel endothelial cells but not in othercell types which have the MuLV/non-MuLV constructs. According to thepresent invention, the modified MuLV LTRs may be used in geneticconstructs useful in gene therapy which are particularly directed atexpressing genes in endothelial cells. Regardless of what other celltypes take up the constructs of the invention, only the vesselendothelial cells which take up the construct will be capable ofexpressing the non-MuLV protein encoded by the sequence operably linkedto the vessel endothelial cell tropic LTR of the invention.

According to some preferred embodiments, the modified MuLV LTR is a MuLVLTR in which nucleotides 131-200 of the LTR are SEQ ID NO:4. Accordingto some preferred embodiments, the modified MuLV LTR is a Friend MuLVLTR in which nucleotides 131-200 of the LTR are SEQ ID NO:4.

According to some preferred embodiments, the modified MuLV LTR is a MuLVLTR in which nucleotides 101-200 of the LTR are SEQ ID NO:3. Accordingto some preferred embodiments, the modified MuLV LTR is a Friend MuLVLTR in which nucleotides 101-200 of the LTR are SEQ ID NO:3.

According to some preferred embodiments, the modified MuLV LTR is a MuLVLTR in which the U3 region is SEQ ID NO:2. According to some preferredembodiments, the modified MuLV LTR is a Friend MuLV LTR in which the U3region is SEQ ID NO:2.

According to some preferred embodiments, the modified MuLV LTR is a MuLVLTR in which nucleotides 1-415 of the LTR are SEQ ID NO:2. According tosome preferred embodiments, the modified MuLV LTR is a Friend MuLV LTRin which nucleotides 1-415 of the LTR are SEQ ID NO:2.

According to some preferred embodiments, the modified MuLV LTR is amodified Friend MuLV LTR having SEQ ID NO:1. SEQ ID NO:1 is the LTR fromthe molecular clone TR1.3, which is a Friend MuLV.

LTRs of retroviruses function as both a promoter/enhancer element aswell as a polyadenylation signal. Accordingly, LTRs may be used upstreamand downstream of the coding sequence to provide the necessaryregulatory sequences for transcription. According to the presentinvention, LTRs are operably linked upstream and/or downstream of thecoding sequence. In genetic contructs which are delivered as linearconstructs, the construct contains a modified LTR sequence of theinvention upstream of and operably linked to a coding sequence thatencodes a non-MuLV protein. In genetic contructs which are delivered ascircular constructs, the construct contains a modified LTR sequence ofthe invention downstream of and operably linked to a coding sequencethat encodes a non-MuLV protein. In both linear and circular constructs,the modified LTR of the invention as insered is operably linked to andupstream of the a coding sequence that encodes a non-MuLV protein. Thoseof ordinary skill in the art can freadily design vectors and constructswhereby the modified LTR of the invention is operably linked to andupstream of the a coding sequence that encodes a non-MuLV protein whenthe non-MuLV protein is being expressed tropically in vessel endothelialcells.

According to some preferred embodiments, the modified MuLV LTR is linkedto the coding sequence that encodes a non-MuLV protein at a location 3'of the coding sequence. According to some preferred embodiments, twomodified MuLV LTRs are linked to the coding sequence that encodes anon-MuLV protein; one at a location 3' of the coding sequence, and oneat a location 5' of the coding sequence.

SEQ ID NO:1 contains a nucleotide sequence of a modified MuLV LTR,specifically the LTR from MuLV TR1.3. This nucleotide sequence ispresented as a cDNA sequence of the genomic RNA of the virus. Themodified LTR disclosed in SEQ ID NO:1 lacks three binding sites, FVb1,FVa and FVb2, as compared with the LTR of wild type Friend MuLV whichreplicates poorly or not at all in endothelial cells. The modified LTRsaccording to the present invention include MuLV LTRs which containdeletions, insertions and substitutions and which are capable ofregulating endothelial cell specific expression of coding sequencesoperably linked thereto. Examples include SEQ ID NO:1. Modified LTRsaccording to the present invention include fragments of SEQ ID NO:1which are capable of regulating endothelial cell specific expression ofcoding sequences operably linked thereto. SEQ ID NO:2 providesnucleotides 1-415 of SEQ ID NO:1. This sequence is the U3 region of theLTR and contains the portion of the LTR which is responsible for thevessel endothelial cell tropism which render the LTR particularlyuseful. SEQ ID NO:3 provides nucleotides 101-200 of SEQ ID NO:1. Thissequence contains the portion of the LTR which is responsible for thevessel endothelial cell tropism which render the LTR particularlyuseful. SEQ ID NO:4 provides nucleotides 131-200 of SEQ ID NO:1. Thissequence contains the portion of the LTR which is responsible for thevessel endothelial cell tropism which render the LTR particularlyuseful.

One having ordinary skill in the art can determine whether or not amodified MuLV LTR is capable of vessel endothelial cell tropicexpression of coding sequences operably linked thereto using standardtechniques and readily available starting materials without undueexperimentation. For example, the nucleic acid molecule can beconstructed that comprises modified LTRs operably linked to a reportergene. A reporter gene is a gene which encodes a protein product that canbe detected. Examples of common reporter genes includebeta-galactosidase or chloramphenicol acetyl transferase. Simple assaysare available to detect the presence of the protein product of thesereporter genes. The nucleic acid molecule is transfected intoendothelial cells and detection of the reporter gene product indicatesthat the LTRs are functioning in the cells. The nucleic acid molecule istransfected into non-endothelial cells. If the reporter gene product isnot detected, the LTRs are not functioning in those cells. Primaryendothelial cell cultures are best suited for in vitro expression offoreign genes. These cultures are easily obtained from human umbilicalcord using standard techniques.

Modified LTRs, such as the modified LTR disclosed in SEQ ID NO:1, or amodified MuLV which comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, ora functional fragment thereof, may be operably linked to nucleotidesequences which encode non-MuLV proteins to form a functional chimericgene. When introduced into endothelial cells, the chimeric gene can beexpressed to produce the protein in the endothelial cells. Inparticular, one or two copies of a modified MuLV LTRs such as that whichis disclosed in SEQ ID NO:1 or a modified MuLV which comprises SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4 or a functional fragment thereof may beoperably linked to a nucleotide sequence that encodes a human protein inorder to express the human protein in human vessel endothelial cells. Insome embodiments, one copy is provided and it is linked upstream of thecoding sequence. In some embodiments, one copy is provided and it islinked downstream of the coding sequence. In some embodiments, twocopies are provided, one is linked upstream of the coding sequence andone copy is linked downstream.

In addition to a vessel endothelial tropic modified MuLV LTR operablelinked to a coding sequence encoding a non-MuLV protein, gene constructsaccording to the invention may additionally comprise nucleotidesequences encoding signal sequences and other amino acid sequenceslinked to the non-MuLV protein sequence to direct secretion orintracellular location of the protein. Signal sequences which directsecretion of protein by endothelial cells are well known and thosehaving ordinary skill in the art can readily design gene construct toexpress non-MuLV proteins in vessel endothelial cells by placing thecoding sequence under the regulatory control of a modified MuLV LTR andfurther directing secretion of the non-MuLV protein so expressed in thevessel endothelial cells. Secretion by vessel endothelial cells can bespecifically directed to result in luminal or abluminal secretion.Further, gene constructs can be designed so that the non-MuLV proteincontains a transmembrane amino acid sequence. Such proteins will beanchored into the cell membrane of the vessel endothelial cell.

In some embodiments, gene constructs provide for delivering codingsequences that encode non-MuLV proteins into primary cells to expresssuch proteins in primary vessel endothelial cells only. In someembodiments, the non-MuLV protein confers antibiotic resistance.Delivery of the gene construct to primary cell cultures results inexpression of the non-MuLV protein in vessel endothelial cells only.Culturing the cells in selection medium, i.e. medium that contains theantibiotic will result in the death of all cells except those expressingthe resistance gene. Thus, only the vessel endothelial cells willsurvive and the resulting culture will be pure primary vesselendothelial cells. The present invention is thus useful for expressingproteins in primary vessel endothelial cells and in some embodiments,for selectively culturing such cells. Transport and secretion ofproteins in endothelial cells and the signals and peptide domainsassociated therewith are discussed in Rodriguez-Boulan, E. and C.Zurzolo 1993 J. Cell Sci. Suppl. 17:9-12 and Rodriguez-Boulan, E. and S.K. Powell 1992 Annu. Rev. Cell Biol. 8:395-427, each of which isincorporated herein by reference.

In some embodiments, construct may be useful in gene therapyapplications in which gene expression and protein production in vesselendothelial cells is desired. The non-MuLV proteins which may beproduced in endothelial cells according to the present invention includehuman and non-human proteins. Examples of human proteins which may beproduced in endothelial cells according to the present inventioninclude, but are not limited to Factor VII protein, Factor IX protein,von Willdebrand factor, complement proteins, insulin, cytokines, tissueplasminogen activator, alpha-L-iduronidase, iduronate sulfatase,heparin, N-sulfatase and alpha 1 antitrypsin. Factor VII protein isuseful to treat individuals suffering from Hemophilia A. Factor IXprotein is useful to treat individuals suffering from Hemophilia B. vonWilldebrand factor is useful to treat individuals suffering from vonWilldebrand's disease. Complement proteins are useful to treatindividuals suffering from complement related immunodeficiencies.Insulin is useful to treat individuals suffering from diabetes.Cytokines are useful to treat individuals suffering from cancer, HIVinfection and hereditary anemias among other conditions. Tissueplasminogen activator is useful in fibrinolytic therapy and in theprevention of stroke due to emboli. Alpha-L-iduronidase, iduronatesulfatase, heparin, N-sulfatase are useful to treat individualssuffering from mucopolysacharidoses and CNS and related disorders. Alpha1 antitrypsin is useful to treat individuals suffering from alpha 1antitrypsin deficiency and lung and related disorders.

According to some preferred embodiments, the coding sequence for humannerve growth factor is provided in gene construct. Delivery of the geneconstruct results in production of human NGF in vessel endothelialcells, facilitating renervation when delivered at or near wound sites.

According to some preferred embodiments, the coding sequence for humanFactor IX is provided in gene construct. Delivery of the gene construct,particularly intravenously to individual suffering from hemophilia,results in production of Factor IX in vessel endothelial cells. Signalsequences may be provided to direct the Factor IX thus expressed to besecreted. Secretion of the Factor IX protein provides the individualwith the necessary proteins for effective clotting.

According to other contemplated embodiments, nucleotide sequencesencoding proteases, digestive enzymes and the like delivered areprovided in gene construct. Delivery of the gene construct, particularlyintravenously to an individual suspected to be at risk of suffering fromor being susceptible to vessel occlusion results in production of theproteins in vessel endothelial cells. Transmembrane sequences may beprovided to direct the location of the protein as being anchored in theblood vessel wall. The protein provides the individual with thenecessary proteins to effectively prevent or reduce occlusion.

The ability to construct nucleic acid molecules, including the genomesof vectors, that exhibit tissue specific tropism will greater facilitatethe applicability of human gene therapy. The ability to constructnucleic acid molecules, including the genomes of vectors, that can betranscriptional active in vessel endothelial cells exclusively areparticularly useful for delivering proteins systemically throughvascular vessel endothelial tissue or proteins directed at hematologicalor vascular disorders.

According to the present invention, vessel endothelial cells can be usedto produce proteins while other cell types in which the gene constructsare also delivered do not express the protein. Vessel endothelial cellsin the brain are particularly useful for expressing chimeric genes whichcomprise modified MuLV LTRs according to the invention.

According to one aspect of the present invention, non-MuLV proteins maybe produced in vessel endothelial cells by introducing into the vesselendothelial cells a chimeric gene which includes a modified LTR operablylinked to a nucleotide sequence which encodes the non-MuLV proteinoperably linked to a modified LTR. Introduction of the chimeric geneinto the vessel endothelial cell will result in the expression of thenucleotide sequence that encodes the non-MuLV protein and, accordingly,the production of the non-MuLV protein.

According to some aspects of the present invention, the human vesselendothelial cells are targeted for production of non-MuLV proteins thatare useful as therapeutics. In particular, the chimeric gene whichincludes modified LTRs operably linked to the nucleotide sequence thatencodes the non-MuLV protein is delivered to the vessel endothelialcells of an individual by way of a vector or other vehicle.

For example, the chimeric gene may be part of a viral vector genome.Such viral vector genomes include those of retroviruses and DNA viruseswhich comprise the chimeric gene. An example of a viral vector system isMiller et al. 1993 Methods of Enzymol. 217:581-599, which isincorporated herein by reference. The nucleic acid sequence of suchviral vectors are encapsulated within a viral particle made up of viralproteins. The viral particle is used to facilitate entry of the nucleicacid molecule that comprises the chimeric gene into the endothelialcell. Once inside the cell, the chimeric gene is expressed and theprotein is produced.

Other methods of introducing nucleic acid molecules into cells arewell-known to those having ordinary skill in the art. For example,nucleic acid molecules encapsulated within liposomes may be used tofacilitate entry of the nucleic acid molecule into an endothelial cells.Liposomes are particularly useful because they have been shown tospecifically target endothelial cells for the delivery of nucleic acidmolecules encapsulated therein. Accordingly, the combination ofliposomes with the chimeric gene comprises a modified MuLV LTR isparticularly useful for delivering chimeric nucleic acid molecules toendothelial cells and expressing the coding sequences of chimeric genes.The production of liposome encapsulated gene constructs and thereadministration in vivo as well as the specific utility of liposomes fordelivering nucleic acid molecules to endothelial cells has been pointedout in Zhu, N. et al. (1993) SCIENCE 261:209-211, which is incorporatedherein by reference. Additionally, Debs, R. J. et al. (1990) J. Biol.Chem. 265(18):10189-10192, which has been incorporated herein byreference, teaches liposome production and administration of liposomeencapsulated compositions.

The gene constructs may be prepared as plasmid DNA, linear DNA or RNA,as part of viral genomes, or as nucleic acid, molecules within viralpackages. Viral packaging is well known and refers to a particular typeof viral vector which is a infectious, non-replicating agent comprisinga nucleic acid molecule such as a gene construct within a viral coat.Viral packages thus provide a means of delivery gene constructs intocells by providing viral-like particles which attach and introducenucleic acid molecules into cells. However, instead of delivering aviral genome capable of directing viral replication, the nucleic acidmolecule delivered by the particle is a gene construct which, in thepresent invention, encodes a non-MuLV protein whose expression isdirected and regulated by the modified MuLV LTR. In packaging systems,cells express the proteins which make up the viral coat and additionallycomprise genetic information to replicate the gene construct. The copiesof the gene construct which are produced are packaged into the viralcoat, yielding particles useful to deliver the gene construct into cellsof an individual. However, once introduced into the cells, the particlescannot replicate. Examples of viral packaging systems include Markowitzet al. 1988 J. Virol. 62:1120-1124, which is incorporated herein byreference.

In some preferred embodiments, the gene construct is packaged in anamphotropic line. An amphotropic line will deliver gene constructs intoall cells. The nature of viral coats is that they require a cellularreceptor for which to attach. Amphotropic packaging lines produce viralparticles which can attach to most or all cells. In some preferredembodiments, the gene construct is packaged in an amphotropic line inwhich the glycoprotein and env proteins of AM12 are expressed.

In some preferred embodiments, the gene constructs are administeredintravenously. In some preferred embodiments, the gene constructs arepackaged in an amphotropic line and delivered intravenously, In somepreferred embodiments, 10⁴ to 10⁶ viral package particles areadministered to an individual. In some preferred embodiments, about 10⁵viral package particles are administered to an individual.

In one embodiment of the present invention a chimeric gene isconstructed with comprises the modified MuLV LTR as disclosed in SEQ IDNO:1 operably linked to a nucleic acid sequence which encodes humanFactor VII protein. The nucleic acid sequence which encodes human FactorVII protein is disclosed in O'Hara, P. J.. et al. (1987) Proc. Natl.Acad. Sci. USA 84:5158-5162, which is incorporated herein by reference.

In one embodiment of the present invention a chimeric gene isconstructed with comprises the modified MuLV LTR as disclosed in SEQ IDNO:1 operably linked to a nucleic acid sequence which encodes humanFactor IX protein. The nucleic acid sequence which encodes human FactorIX protein is disclosed in Yao, S. et al. (1991) Proc. Natl. Acad. Sci.USA 88:8101-8105, which is incorporated herein by reference.

In some embodiments of the invention, the chimeric gene is inserted intoretroviral vectors using a complementation system using vectors such asSV-psi⁻ -env⁻ -MLV and SV-psi⁻ -A-MLV. These vectors are disclosed inLandau, N. R. and D. R. Littman (1992) J. Virol. 66(8):5110-5114, whichis incorporated herein by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 574 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATGAAAGACCCCACCAAATTGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGC60                ATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACACGAAAACA120               GCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAA180               GAACAGATGGTCACCGCAGTTCGGCCCCGGCCCGGGCGAAGAACAGATGGTCCCCAGATA240               TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGAC300               CTGAAATGACCCTGTGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCG360               CGCGCTTCTGCTTCCCTAGCCCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGT420               CCTCCGACAGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAATCCTCTTGCTGTTGCA480               TCCGACTCGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCAGAGTGATTGACTACCCGTC540               TCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGAT574                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 415 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AATGAAAGACCCCACCAAATTGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGC60                ATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACACGAAAACA120               GCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAA180               GAACAGATGGTCACCGCAGTTCGGCCCCGGCCCGGGCGAAGAACAGATGGTCCCCAGATA240               TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGAC300               CTGAAATGACCCTGTGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCG360               CGCGCTTCTGCTTCCCTAGCCCTATAAAAGAGCTCACAACCCCTCACTCGGCGCG415                    (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGGGCGGGTACACGAAAACAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTT60                TCGGCCCCGGCCCGGGCGAAGAACAGATGGTCACCGCAGT100                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGCGAAGAACAGATGG60                TCACCGCAGT70                                                                  __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule comprising a modifiedMurine Leukemia Virus Long Terminal Repeat which comprises SEQ ID NO:4.2. The nucleic acid molecule of claim 1 wherein said modified MurineLeukemia Virus Long Terminal Repeat sequence comprises SEQ ID NO:3. 3.The nucleic acid molecule of claim 1 wherein said modified MurineLeukemia Virus Long Terminal Repeat sequence comprises SEQ ID NO:2. 4.The nucleic acid molecule of claim 1 wherein said modified MurineLeukemia Virus Long Terminal Repeat sequence comprises SEQ ID NO:1. 5.The nucleic acid molecule of claim 1 wherein said modified MurineLeukemia Virus Long Terminal Repeat sequence comprises SEQ ID NO:4 andis operably linked to a nucleotide sequence that encodes a non-MurineLeukemia Virus protein.
 6. The nucleic acid molecule of claim 1 whereinsaid modified Murine Leukemia Virus Long Terminal Repeat sequencecomprises SEQ ID NO:3 and is operably linked to a nucleotide sequencethat encodes a non-Murine Leukemia Virus protein.
 7. The nucleic acidmolecule of claim 1 wherein said modified Murine Leukemia Virus LongTerminal Repeat sequence comprises SEQ ID NO:2 and is operably linked toa nucleotide sequence that encodes a non-Murine Leukemia Virus protein.8. The nucleic acid molecule of claim 1 wherein said modified MurineLeukemia Virus Long Terminal Repeat Sequence comprises SEQ ID NO:1 andis operably linked to a nucleotide sequence that encodes a non-MurineLeukemia Virus protein.
 9. The nucleic acid molecule of claim 5 whereinsaid non-Murine Leukemia Virus protein is a human protein.
 10. Thenucleic acid molecule of claim 5 wherein said non-Murine Leukemia Virusprotein is a human protein selected from the group consisting of: FactorVII protein, Factor IX protein, von Willdebrand factor, complementproteins, insulin, cytokines, tissue plasminogen activator,alpha-L-iduronidase, iduronate sulfatase, heparin, N-sulfatase and alpha1 antitrypsin.
 11. The nucleic acid molecule of claim 5 wherein saidnucleic acid molecule is encapsulated within a liposome or a viral coat.12. The nucleic acid molecule of claim 11 wherein said nucleic acidmolecule is encapsulated within a liposome.
 13. The nucleic acidmolecule of claim 5 wherein said nucleic acid molecule is encapsulatedwithin a viral coat.
 14. The nucleic acid molecule of claim 13 whereinsaid nucleic acid molecule is incorporated within a viral genome. 15.The nucleic acid molecule of claim 13 wherein said nucleic acid moleculeis incorporated with a retroviral genome.
 16. The nucleic acid moleculeof claim 13 wherein said nucleic acid molecule is encapsulated in aninfectious, non-replicating virus package particle.
 17. A method ofproducing a non-Murine Leukemia Virus protein in an endothelial cellthat comprises the step of:introducing into said endothelial cell,nucleic acid molecule comprising a modified Murine Leukemia Virus LongTerminal Repeat which comprises SEQ ID NO:4 operably linked to anucleotide sequence that encodes a non-Murine Leukemia Virus protein.18. The method of claim 17 wherein said cell is a human vesselendothelial cell.
 19. The method of claim 17 wherein said modifiedMurine Leukemia Virus Long Terminal Repeat sequence comprises SEQ IDNO:3.
 20. The method of claim 17 wherein said modified Murine LeukemiaVirus Long Terminal Repeat sequence comprises SEQ ID NO:2.
 21. Themethod of claim 17 wherein said modified Murine Leukemia Virus LongTerminal Repeat sequence comprises SEQ ID NO:1.
 22. The method of claim17 wherein said non-Murine Leukemia Virus protein is a human protein.23. The method of claim 17 wherein said non-Murine Leukemia Virusprotein is a human protein selected from the group consisting of FactorVII protein, Factor IX protein, von Willdebrand factor, complementproteins, insulin, cytokines, tissue plasminogen activator,alpha-L-iduronidase, iduronate sulfatase, heparin, N-sulfatase and alpha1 antitrypsin.
 24. The method of claim 17 wherein said nucleic acidmolecule is encapsulated within a liposome or a viral coat.
 25. Themethod of claim 11 wherein said nucleic acid molecule is encapsulatedwithin a viral coat of a retrovirus particle.
 26. The method of claim 11wherein said nucleic acid molecule is encapsulated within a viral coatof an infectious, non-replicating viral package particle.